From 2011.igem.org

Redesigning Kumamolisin to Have Higer Activity at Low pH

Using Foldit to Produce Mutations

In order to create mutations from wild-type kumamolisin, we first loaded the enzyme structure into the protein editing game Foldit. Foldit allows the user to modify the way in which a protein folds by changing the amino acid sequence. Using Foldit, we modified the amino acid residues around the active site of kumamolisin which contained within it a Proline-Glutamine-Lysine-Proline (PQLP) motif found throughout the gliadin peptide of gluten. With the PQLP locked in place along with the key active site amino acid residues, mutations were made which appeared to stabilize PQLP within the active site. Using this method, we produced several mutants at a time which could potentially increase the enzyme's proteolytic activity towards gliadin.

Mutagenizing Kumamolisin

Using a Whole Cell Lysate Assay to Test Feasability of Mutant

Purifying Mutants to Accurately Assess Activity

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